Changes in the structure and function of the Nicotinic Acetyicholine Receptor (AChR) are linked to pathogenic responses on human muscle and brain. The long-term goal of this research proposal is to define the functional role of lipid-protein interactions in the conformational transitions of the ACbR. The objective of this project is to define the modes by which hydrophobic allosteric interactions are linked to the gating machinery of the AChR and bow cholesterol modulates ACbR function. The central hypothesis of this research is that AChR channel kinetics is modulated allosterically by specific sites on the receptor that are in direct contact with the lipid-interface. This hypothesis has been formulated on the basis of strong preliminary data, which suggest that: (1) single amino acid replacements at hydrophobic allosteric sites of the M3 and M4 iransmembrane segments of the AChR greatly enhance the macroscopic response to acetyicholine and produce a remarkable alteration of the modulatory role of cholesterol on the AChR function, and (2) cholesterol plays a novel role in the transition of AChRs from silent to functional membrane pools. The rationale for the proposed research is that changes in membrane lipid composition (and lipid-protein interactions) represent a very important mechanism for the regulation of AChR channel function in cholinergic synapses of muscle and brain, thus understanding the molecular basis of these mechanisms will help to identify conditions in which the membrane lipid composition enhance or inhibit AChR function. The central hypothesis will be tested by pursuing four specific aims: (1) To study the structure-function relationship of five hydrophobic allosteric positions using additional side chain replacements, (2) Introduce periodic tryptophan substitutions to complete all positions along the M3 transmembrane domains in order to define helix-helix contacts, structural constraint positions and additional allosteric sites, (3) Examine the effects of membrane cholesterol levels on AChR channel kinetics and five new hydrophobic ailosteric M3 mutations and (4) Introduce unnatural amino acids at hydrophobic allosteric positions in the M3 domain to determine; (a) the effect of electronic density on channel gating properties and (b) membrane penetration depth of the allosteric positions. The proposed work is innovative because it capitalizes on new approaches, developed by the applicants, to; 1) perform cholesterol enrichment and depletions in the plasmatic membrane of oocytes, and 2) introduce fluorescent unnatural amino acid to assess membrane depth penetration and annular lipid composition using multiphoton confocal imaging. We anticipate that these approaches will allow us to identify new mechanisms by which lipid-protein interactions modulate the allosteric transitions of AChR. The knowledge gained from these studies is of paramount significance as it will: 1) describe new mechanisms for AChR regulation in the intact synapse, 2) provide information on the secondary structure and spatial organization of the M3 and M4 domains, and 3) describe new structure-function relationships for the AChR.